Functional analysis of CLIP-115 and its binding to microtubules

2000 ◽  
Vol 113 (12) ◽  
pp. 2285-2297 ◽  
Author(s):  
C.C. Hoogenraad ◽  
A. Akhmanova ◽  
F. Grosveld ◽  
C.I. De Zeeuw ◽  
N. Galjart
Keyword(s):  
Functional Analysis ◽  
Coiled Coil ◽  
Single Domain ◽  
Disulfide Bridges ◽  
Microtubule Network ◽  
Microtubule Binding ◽  
Neuronal Dendrites ◽  
Cytoskeletal Network ◽  
Coil Structure

Cytoplasmic linker proteins (CLIPs) bind to microtubules and are proposed to link this cytoskeletal network to other intracellular structures. We are interested in CLIP-115, since this protein is enriched in neuronal dendrites and may operate in the control of brain-specific organelle translocations. Each CLIP monomer is characterized by two microtubule-binding (MTB) motifs, surrounded by basic, serine-rich regions. This head domain is connected to the C-terminal tail through a long coiled-coil structure. The MTB domains are conserved as a single domain in other proteins involved in microtubule based transport and dynamics, such as p150(Glued). Here we provide evidence that efficient binding of CLIP-115 to microtubules is sensitive to phosphorylation and is not mediated by the conserved MTB domains alone, but requires the presence of the basic, serine rich regions in addition to the MTB motifs. In transfected COS-1 cells, CLIP-115 initially accumulates at the distal ends of microtubules and coincides with CLIP-170, indicating that both proteins mark growing microtubule ends. However, when expressed at higher levels, CLIP-115 and -170 affect the microtubule network differently. This might be partly due to the divergent C-termini of the two proteins. We demonstrate that, similar to CLIP-170, CLIP-115 forms homodimers, which, at least in vitro, are linked by disulfide bridges. Cysteine(391) of CLIP-115, however, is specific in that it controls the microtubule bundling capacity of certain mutant CLIP-115 molecules. Therefore, both similar and specific mechanisms appear to regulate the conformation of CLIPs as well as their binding to microtubules.

scholarly journals MTCL2 is a new Golgi-resident microtubule-regulating protein, essential for organizing asymmetric microtubule network

2020 ◽  
Author(s):  
Risa Matsuoka ◽  
Masateru Miki ◽  
Sonoko Mizuno ◽  
Yurina Ito ◽  
Atsushi Suzuki
Keyword(s):  
Coiled Coil ◽  
Golgi Membrane ◽  
Microtubule Network ◽  
Microtubule Binding ◽  
Microtubule Stabilization ◽  
Binding Domains ◽  
Microtubule Binding Domain ◽  
Ribbon Structure ◽  
Golgi Ribbon

AbstractThe Golgi apparatus plays important roles in organizing the asymmetric microtubule network essential for polarized vesicle transport. The Golgi-associated coiled-coil protein MTCL1 is crucially involved in Golgi functioning by interconnecting and stabilizing microtubules on the Golgi membrane through its N- and C-terminal microtubule-binding domains. Here, we report the presence of a mammalian paralog of MTCL1, named MTCL2, lacking the N-terminal microtubule-binding domain. MTCL2 localizes to the Golgi membrane through the N-terminal region and directly binds microtubules through the conserved C-terminal domain without promoting microtubule stabilization. Knockdown experiments demonstrated essential roles of MTCL2 in accumulating MTs around the Golgi and regulating the Golgi ribbon structure. In vitro wound healing assays further suggested a possible intriguing activity of MTCL2 in integrating the centrosomal and Golgi-associated microtubules around the Golgi ribbon, thus supporting directional migration. Altogether, the present results demonstrate that cells utilize two members of the MTCL protein family to differentially regulate the Golgi-associated microtubules for controlling cell polarity.

scholarly journals GMAP-210, A Cis-Golgi Network-associated Protein, Is a Minus End Microtubule-binding Protein

1999 ◽  
Vol 145 (1) ◽  
pp. 83-98 ◽  
Author(s):  
Carlos Infante ◽  
Francisco Ramos-Morales ◽  
Concepción Fedriani ◽  
Michel Bornens ◽  
Rosa M. Rios
Keyword(s):  
Golgi Apparatus ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Coiled Coil ◽  
Microtubule Network ◽  
Microtubule Binding ◽  
Terminal Domain ◽  
Green Fluorescent ◽  
Golgi Network

We report that a peripheral Golgi protein with a molecular mass of 210 kD localized at the cis-Golgi network (Rios, R.M., A.M. Tassin, C. Celati, C. Antony, M.C. Boissier, J.C. Homberg, and M. Bornens. 1994. J. Cell Biol. 125:997–1013) is a microtubule-binding protein that associates in situ with a subpopulation of stable microtubules. Interaction of this protein, now called GMAP-210, for Golgi microtubule-associated protein 210, with microtubules in vitro is direct, tight and nucleotide-independent. Biochemical analysis further suggests that GMAP-210 specifically binds to microtubule ends. The full-length cDNA encoding GMAP-210 predicts a protein of 1,979 amino acids with a very long central coiled-coil domain. Deletion analyses in vitro show that the COOH terminus of GMAP-210 binds to microtubules whereas the NH2 terminus binds to Golgi membranes. Overexpression of GMAP-210–encoding cDNA induced a dramatic enlargement of the Golgi apparatus and perturbations in the microtubule network. These effects did not occur when a mutant lacking the COOH-terminal domain was expressed. When transfected in fusion with the green fluorescent protein, the NH2-terminal domain associated with the cis-Golgi network whereas the COOH-terminal microtubule-binding domain localized at the centrosome. Altogether these data support the view that GMAP-210 serves to link the cis-Golgi network to the minus ends of centrosome-nucleated microtubules. In addition, this interaction appears essential for ensuring the proper morphology and size of the Golgi apparatus.

scholarly journals A Structural Model for Phosphorylation Control of Dictyostelium Myosin II Thick Filament Assembly

1999 ◽  
Vol 147 (5) ◽  
pp. 1039-1048 ◽  
Author(s):  
Wenchuan Liang ◽  
Hans M. Warrick ◽  
James A. Spudich
Keyword(s):  
Structural Model ◽  
Myosin Ii ◽  
Coiled Coil ◽  
Thick Filament ◽  
Tail Region ◽  
Filament Assembly ◽  
Dictyostelium Myosin ◽  
Thick Filaments ◽  
Coil Structure

Myosin II thick filament assembly in Dictyostelium is regulated by phosphorylation at three threonines in the tail region of the molecule. Converting these three threonines to aspartates (3×Asp myosin II), which mimics the phosphorylated state, inhibits filament assembly in vitro, and 3×Asp myosin II fails to rescue myosin II–null phenotypes. Here we report a suppressor screen of Dictyostelium myosin II–null cells containing 3×Asp myosin II, which reveals a 21-kD region in the tail that is critical for the phosphorylation control. These data, combined with new structural evidence from electron microscopy and sequence analyses, provide evidence that thick filament assembly control involves the folding of myosin II into a bent monomer, which is unable to incorporate into thick filaments. The data are consistent with a structural model for the bent monomer in which two specific regions of the tail interact to form an antiparallel tetrameric coiled–coil structure.

scholarly journals Coronin Promotes the Rapid Assembly and Cross-linking of Actin Filaments and May Link the Actin and Microtubule Cytoskeletons in Yeast

1999 ◽  
Vol 144 (1) ◽  
pp. 83-98 ◽  
Author(s):  
Bruce L. Goode ◽  
Jonathan J. Wong ◽  
Anne-Christine Butty ◽  
Matthias Peter ◽  
Ashley L. McCormack ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Coiled Coil ◽  
Cross Linking ◽  
Cortical Actin ◽  
Functional Link ◽  
Microtubule Binding ◽  
Filament Assembly ◽  
Cell Extracts ◽  
Cross Links

Coronin is a highly conserved actin-associated protein that until now has had unknown biochemical activities. Using microtubule affinity chromatography, we coisolated actin and a homologue of coronin, Crn1p, from Saccharomyces cerevisiae cell extracts. Crn1p is an abundant component of the cortical actin cytoskeleton and binds to F-actin with high affinity (Kd 6 × 10−9 M). Crn1p promotes the rapid barbed-end assembly of actin filaments and cross-links filaments into bundles and more complex networks, but does not stabilize them. Genetic analyses with a crn1Δ deletion mutation also are consistent with Crn1p regulating filament assembly rather than stability. Filament cross-linking depends on the coiled coil domain of Crn1p, suggesting a requirement for Crn1p dimerization. Assembly-promoting activity is independent of cross-linking and could be due to nucleation and/or accelerated polymerization. Crn1p also binds to microtubules in vitro, and microtubule binding is enhanced by the presence of actin filaments. Microtubule binding is mediated by a region of Crn1p that contains sequences (not found in other coronins) homologous to the microtubule binding region of MAP1B. These activities, considered with microtubule defects observed in crn1Δ cells and in cells overexpressing Crn1p, suggest that Crn1p may provide a functional link between the actin and microtubule cytoskeletons in yeast.

scholarly journals Sequences in the stalk domain regulate autoinhibition and ciliary tip localization of the immotile kinesin-4 KIF7

2021 ◽  
Author(s):  
T. Lynne Blasius ◽  
Yang Yue ◽  
Raghu Ram Prasad ◽  
Xinglei Liu ◽  
Arne Gennerich ◽  
...  
Keyword(s):  
Primary Cilium ◽  
Human Disease ◽  
Hedgehog Signaling ◽  
Coiled Coil ◽  
Full Length ◽  
Microtubule Binding ◽  
Stalk Domain ◽  
In Cells

The kinesin-4 member KIF7 plays critical roles in Hedgehog signaling in vertebrate cells. KIF7 is an atypical kinesin as it binds to microtubules but is immotile. We demonstrate that, like conventional kinesins, KIF7 is regulated by autoinhibition as the full-length protein is inactive for microtubule binding in cells. We identify a segment, the inhibitory coiled coil (inhCC), that is required for autoinhibition of KIF7 whereas the adjacent regulatory coiled coil (rCC) that contributes to autoinhibition of the motile kinesin-4's KIF21A and KIF21B, is not sufficient for KIF7 autoinhibition. Disease-associated mutations in the inhCC relieve autoinhibition and result in strong microtubule binding. Surprisingly, uninhibited KIF7 proteins did not bind preferentially to or track the plus ends of growing microtubules in cells, as suggested by previous in vitro work, but rather bound along cytosolic and axonemal microtubules. Localization to the tip of the primary cilium also required the inhCC and could be increased by disease-associated mutations regardless of the protein's autoinhibition state. These findings suggest that loss of KIF7 autoinhibition and/or altered cilium tip localization can contribute to pathogenesis of human disease.

scholarly journals A microtubule-binding protein associated with membranes of the Golgi apparatus.

1986 ◽  
Vol 103 (6) ◽  
pp. 2229-2239 ◽  
Author(s):  
V J Allan ◽  
T E Kreis
Keyword(s):  
Golgi Apparatus ◽  
Culture Cell ◽  
Proteinase K ◽  
Microtubule Network ◽  
Microtubule Binding ◽  
Golgi Stack ◽  
Cell Extracts ◽  
Triton X

A monoclonal antibody (M3A5), raised against microtubule-associated protein 2 (MAP-2), recognized an antigen associated with the Golgi complex in a variety of non-neuronal tissue culture cells. In double immunofluorescence studies M3A5 staining was very similar to that of specific Golgi markers, even after disruption of the Golgi apparatus organization with monensin or nocodazole. M3A5 recognized one band of Mr approximately 110,000 in immunoblots of culture cell extracts; this protein, designated 110K, was enriched in Golgi stack fractions prepared from rat liver. The 110K protein has been shown to partition into the aqueous phase by Triton X-114 extraction of a Golgi-enriched fraction and was eluted after pH 11.0 carbonate washing. It is therefore likely to be a peripheral membrane protein. Proteinase K treatment of an isolated Golgi stack fraction resulted in complete digestion of the 110K protein, both in the presence and absence of Triton X-100. A the 110K protein is accessible to protease in intact vesicles in vitro, it is presumably located on the cytoplasmic face of the Golgi membrane in vivo. The 110K protein was able to interact specifically with taxol-polymerized microtubules in vitro. These results suggest that the 110K protein may serve to link the Golgi apparatus to the microtubule network and so may belong to a novel class of proteins: the microtubule-binding proteins.

scholarly journals C-Nap1, a Novel Centrosomal Coiled-Coil Protein and Candidate Substrate of the Cell Cycle–regulated Protein Kinase Nek2

1998 ◽  
Vol 141 (7) ◽  
pp. 1563-1574 ◽  
Author(s):  
Andrew M. Fry ◽  
Thibault Mayor ◽  
Patrick Meraldi ◽  
York-Dieter Stierhof ◽  
Kayoko Tanaka ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Coiled Coil ◽  
Western Blots ◽  
Interacting Protein ◽  
Calculated Molecular Mass ◽  
Mammalian Cell Cycle ◽  
Coil Structure ◽  
Mother And Daughter

Nek2 (for NIMA-related kinase 2) is a mammalian cell cycle–regulated kinase structurally related to the mitotic regulator NIMA of Aspergillus nidulans. In human cells, Nek2 associates with centrosomes, and overexpression of active Nek2 has drastic consequences for centrosome structure. Here, we describe the molecular characterization of a novel human centrosomal protein, C-Nap1 (for centrosomal Nek2-associated protein 1), first identified as a Nek2-interacting protein in a yeast two-hybrid screen. Antibodies raised against recombinant C-Nap1 produced strong labeling of centrosomes by immunofluorescence, and immunoelectron microscopy revealed that C-Nap1 is associated specifically with the proximal ends of both mother and daughter centrioles. On Western blots, anti–C-Nap1 antibodies recognized a large protein (>250 kD) that was highly enriched in centrosome preparations. Sequencing of overlapping cDNAs showed that C-Nap1 has a calculated molecular mass of 281 kD and comprises extended domains of predicted coiled-coil structure. Whereas C-Nap1 was concentrated at centrosomes in all interphase cells, immunoreactivity at mitotic spindle poles was strongly diminished. Finally, the COOH-terminal domain of C-Nap1 could readily be phosphorylated by Nek2 in vitro, as well as after coexpression of the two proteins in vivo. Based on these findings, we propose a model implicating both Nek2 and C-Nap1 in the regulation of centriole–centriole cohesion during the cell cycle.

Rod mutations associated with MYH9-related disorders disrupt nonmuscle myosin-IIA assembly

Blood ◽  
2005 ◽  
Vol 105 (1) ◽  
pp. 161-169 ◽  
Author(s):  
Josef D. Franke ◽  
Fan Dong ◽  
Wayne L. Rickoll ◽  
Michael J. Kelley ◽  
Daniel P. Kiehart
Keyword(s):  
Coiled Coil ◽  
Chain Gene ◽  
Nonmuscle Myosin ◽  
Wild Type ◽  
Negative Stain ◽  
Coil Structure ◽  
Negative Stain Electron Microscopy ◽  
Myosin Iia ◽  
Mutant Forms

Abstract MYH9-related disorders are autosomal dominant syndromes, variably affecting platelet formation, hearing, and kidney function, and result from mutations in the human nonmuscle myosin-IIA heavy chain gene. To understand the mechanisms by which mutations in the rod region disrupt nonmuscle myosin-IIA function, we examined the in vitro behavior of 4 common mutant forms of the rod (R1165C, D1424N, E1841K, and R1933Stop) compared with wild type. We used negative-stain electron microscopy to analyze paracrystal morphology, a model system for the assembly of individual myosin-II molecules into bipolar filaments. Wild-type tail fragments formed ordered paracrystal arrays, whereas mutants formed aberrant aggregates. In mixing experiments, the mutants act dominantly to interfere with the proper assembly of wild type. Using circular dichroism, we find that 2 mutants affect the α-helical coiled-coil structure of individual molecules, and 2 mutants disrupt the lateral associations among individual molecules necessary to form higher-order assemblies, helping explain the dominant effects of these mutants. These results demonstrate that the most common mutations in MYH9, lesions in the rod, cause defects in nonmuscle myosin-IIA assembly. Further, the application of these methods to biochemically characterize rod mutations could be extended to other myosins responsible for disease.

scholarly journals Interaction between the Caenorhabditis elegans centriolar protein SAS-5 and microtubules facilitates organelle assembly

2018 ◽  
Vol 29 (6) ◽  
pp. 722-735 ◽  
Author(s):  
Sarah Bianchi ◽  
Kacper B. Rogala ◽  
Nicola J. Dynes ◽  
Manuel Hilbert ◽  
Sebastian A. Leidel ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Microtubule Network ◽  
Microtubule Binding ◽  
C Elegans ◽  
Evolutionarily Conserved ◽  
Organelle Assembly ◽  
In Vivo Analysis ◽  
Cilia And Flagella

Centrioles are microtubule-based organelles that organize the microtubule network and seed the formation of cilia and flagella. New centrioles assemble through a stepwise process dependent notably on the centriolar protein SAS-5 in Caenorhabditis elegans. SAS-5 and its functional homologues in other species form oligomers that bind the centriolar proteins SAS-6 and SAS-4, thereby forming an evolutionarily conserved structural core at the onset of organelle assembly. Here, we report a novel interaction of SAS-5 with microtubules. Microtubule binding requires SAS-5 oligomerization and a disordered protein segment that overlaps with the SAS-4 binding site. Combined in vitro and in vivo analysis of select mutants reveals that the SAS-5–microtubule interaction facilitates centriole assembly in C. elegans embryos. Our findings lead us to propose that the interdependence of SAS-5 oligomerization and microtubule binding reflects an avidity mechanism, which also strengthens SAS-5 associations with other centriole components and, thus, promotes organelle assembly.

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